Bacterial-Nanoparticle Interactions

Bionanotechnology is an intersection between biology and nanotechnology, a field in which novel applications for very small materials are being realised at an alarming rate. Nanoparticles have 3 dimensions that can be measured in nanometers, their small size conferring upon them different properties from individual atoms or the bulk material. The interactions between these unique materials and microorganisms are often toxic, thus have been exploited for antimicrobial applications. However, there is a considerable paucity of data for the underlying molecular mechanisms. This study has been carried out to investigate the interactions that occur between nanoparticles and bacteria with the objective of identifying these toxicological mechanisms and novel nanoparticle effects, using the model Gram negative organism Escherichia coli K12. This study has identified metal nanoparticles that are a superior vehicle for the delivery of toxic metal ions to E. coli. The nanoparticles associate with the bacterial surface, but do not cross the cell wall. They then dissolve, releasing a concentration of metal ions that accumulate at the bacterial-nanoparticle interface, enhancing the antibacterial efficacy compared to the concentration of metal ions in the bulk solution phase. Measurement of the whole transcriptome response to silver nanoparticles in comparison to the silver ion indicates that the different modes of ion delivery may induce a differential stress response. Moreover, this data identifies molecular mechanisms that are involved in the toxicity of this metal that is now becoming increasingly prevalent in society. The dissolution based toxic effects of zinc oxide nanoparticles are augmented by an interaction with ultra-violet light, offering an alternative mode for nanoparticle toxicity.BBSRCExeter Nanobacteria Lt

Recombinase polymerase amplification for fast, selective, DNA‐based detection of faecal indicator Escherichia coli

The bacterium Escherichia coli is commonly associated with the presence of faecal contamination in environmental samples, and is therefore subject to statutory surveillance. This is normally done using a culture‐based methodology, which can be slow and laborious. Nucleic acid amplification for the detection of E. coli DNA sequences is a significantly more rapid approach, suited for applications in the field such as a point of sample analysis, and to provide an early warning of contamination. An existing, high integrity qPCR method to detect the E. coli ybbW gene, which requires almost an hour to detect low quantities of the target, was compared with a novel, isothermal RPA method, targeting the same sequence but achieving the result within a few minutes. The RPA technique demonstrated equivalent inclusivity and selectivity, and was able to detect DNA extracted from 100% of 99 E. coli strains, and exclude 100% of 30 non‐target bacterial species. The limit of detection of the RPA assay was at least 100 target sequence copies. The high speed, and simple, isothermal amplification chemistry may indicate that RPA is a more suitable methodology for on‐site E. coli monitoring than an existing qPCR technique

The regulation of copper stress response genes in the Polychaete Nereis diversicolor during prolonged extreme copper contamination

Polychaetes are frequented in toxicological studies, one reason being that some members occupy shallow burrows in sediments and are maximally exposed to the contaminants that accumulate within them. We have been studying one population of the polychaete Nereis (Hediste) diversicolor exhibiting inheritable tolerance to extreme copper contamination in estuarine sediment. Using transcriptome sequencing data we have identified a suite of genes with putative roles in metal detoxification and tolerance, and measured their regulation. Copper tolerant individuals display significantly different gene expression profiles compared to animals from a nearby population living without remarkable copper levels. Gene transcripts encoding principle copper homeostasis proteins including membrane copper ion transporters, copper ion chaperones and putative metallothionein-like proteins were significantly more abundant in tolerant animals occupying contaminated sediment. In contrast, those encoding antioxidants and cellular repair pathways were unchanged. Nontolerant animals living in contaminated sediment showed no difference in copper homeostasis-related gene expression but did have significantly elevated levels of mRNAs encoding Glutathione Peroxidase enzymes. This study represents the first use of functional genomics to investigate the copper tolerance trait in this species and provides insight into the mechanism used by these individuals to survive and flourish in conditions which are lethal to their conspecifics

Investigation of Occupier Influence on Indoor Air Quality in Dwellings

The aim of the research was to investigate dwelling occupant interaction with natural ventilation components and record levels of indoor air quality. The project also identifies options for making natural ventilation in dwellings a robust strategy

Occupant interactions and effectiveness of natural ventilation strategies in contemporary new housing in Scotland, UK

The need to reduce carbon emissions and fuel poverty has led to increased building envelope air tightness, intended to reduce uncontrolled ventilation heat losses. Ventilation strategies in dwellings still allow the use of trickle ventilators in window frames for background ventilation. The extent to which this results in “healthy” Indoor Air Quality (IAQ) in recently constructed dwellings was a concern of regulators in Scotland. This paper describes research to explore this. First a review of literature was conducted, then data on occupant interactions with ventilation provisions (windows, doors, trickle vents) gathered through an interview-based survey of 200 recently constructed dwellings, and measurements made on a sample of 40 of these. The main measured parameter discussed here is CO2 concentration. It was concluded after the literature review that 1000 ppm absolute was a reasonable threshold to use for “adequate” ventilation. The occupant survey found that there was very little occupant interaction with the trickle ventilators e.g., in bedrooms 63% were always closed, 28% always open, and in only 9% of cases occupants intervened to make occasional adjustments. In the measured dwellings average bedroom CO2 levels of 1520 ppm during occupied (night time) hours were observed. Where windows were open the average bedroom CO2 levels were 972 ppm. With windows closed, the combination of “trickle ventilators open plus doors open” gave an average of 1021 ppm. “Trickle ventilators open” gave an average of 1571 ppm. All other combinations gave averages of 1550 to 2000 ppm. Ventilation rates and air change rates were estimated from measured CO2 levels, for all dwellings calculated ventilation rate was less than 8 L/s/p, in 42% of cases calculated air change rate was less than 0.5 ach. It was concluded that trickle ventilation as installed and used is ineffective in meeting desired ventilation rates, evidenced by high CO2 levels reported across the sampled dwellings. Potential implications of the results are discussed

‘Ready Mixed’, improved nucleic acid amplification assays for the detection of Escherichia coli DNA and RNA

The selective amplification of E. coli nucleic acid sequences could be used for the early warning of faecal contamination in environmental samples. Modified assays for E. coli DNA and RNA markers are presented with improved integrity and performance over existing methods, and demonstrated using ‘ready mixed’, preserved reagent mixtures

A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters

The presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination

Differential gene regulation in the Ag nanoparticle and Ag+-induced silver stress response in Escherichia coli: A full transcriptomic profile

We report the whole-transcriptome response of Escherichia coli bacteria to acute treatment with silver nanoparticles (AgNPs) or silver ions [Ag(I)] as silver nitrate using gene expression microarrays. In total, 188 genes were regulated by both silver treatments, 161 were up-regulated and 27 were down-regulated. Significant regulation was observed for heat shock response genes in line with protein denaturation associated with protein structure vulnerability indicating Ag(I)-labile –SH bonds. Disruption to iron–sulphur clusters led to the positive regulation of iron–sulphur assembly systems and the expression of genes for iron and sulphate homeostasis. Further, Ag ions induced a redox stress response associated with large (>600-fold) up-regulation of the E. coli soxS transcriptional regulator gene. Ag(I) is isoelectronic with Cu(I), and genes associated with copper homeostasis were positively regulated indicating Ag(I)-activation of copper signalling. Differential gene expression was observed for the silver nitrate and AgNP silver delivery. Nanoparticle delivery of Ag(I) induced the differential regulation of 379 genes; 309 genes were uniquely regulated by silver nanoparticles and 70 genes were uniquely regulated by silver nitrate. The differential silver nanoparticle–silver nitrate response indicates that the toxic effect of labile Ag(I) in the system depends upon the mechanism of delivery to the target cell

Ability of decentralised mechanical ventilation to act as ‘whole-house’ ventilation systems in new-build dwellings

As improvements to energy standards have led to greater levels of air tightness in buildings to reduce uncontrolled heat loss, a consequence is that the ventilation provision in modern homes now has to be designed. In current building regulations guidance, airtight buildings (between 3 and 5 m3/hr/m2 @50 Pa) require mechanical ventilation. One method for achieving this is through the use of dMEV systems. These are an increasingly popular strategy due to the relatively low cost and ease of integration. However, some concerns have been expressed about the ability of these systems to provide whole house ventilation, particularly in the light of changes to the guidance for trickle vents. To investigate these issues the Scottish Government Building Standards Division (BSD) commissioned a study that would examine the real world performance of modern homes with dMEV systems. The study undertook a survey of 223 homes to ask occupants about their knowledge and operation of their ventilation system, and a subset of these homes were monitored to examine the actual ventilation performance and determine the factors that affect this. A further study was undertaken in a selected dwelling to experiment with different ventilation strategies using dMEV to identify key factors. The survey found that although there was good awareness of the presence of ventilation provisions, there was a lack of knowledge regarding how these systems were controlled. Many households did not know how to boost the ventilation rate in the dMEV system (or didn’t feel the need to do so), and a lack of engagement with trickle vents was clear. The monitoring found that over 50% of homes appeared to have poor ventilation overnight (where carbon dioxide levels exceeded 1,000ppm for the majority of the time), and that bedrooms were a particular cause for concern. There were a number of variables that affected this. These included the nature of the trickle vents, the window coverings, the path between the room and the dMEV (including the door opening or undercut, and the arrangement of the home) and the installation and performance of the system. Essentially homes with shorter, more open paths for air movement performed better, but rooms which relied on more remote dMEV systems frequently had poor ventilation. Inspection of the monitored homes found a high number of installed dMEV systems (42-52% - depending on location) were sub-optimal (exceeding recommended airflow rates by >15%), or non-compliant with the guidance (17-48%). Flow rates were highly variable, sometimes this was due to the system setup and commissioning, but some systems had provision for occupant control. Given that bedroom doors were often closed (41%) due to occupant preference or fire requirements, the strategy relies on door undercuts but these were undersized in 20% of properties. There were a number of homes (51%) where trickle vents were installed in wet rooms with dMEV systems. Whilst this may improve the efficacy of extract and moisture control in these rooms, this undermines the ability of the system to assist with ventilation in more remote rooms. Whilst dMEV systems in ensuite bathrooms provided the best outcomes for adjacent bedrooms, problems with systems being disabled were encountered in 56% of homes and the predominate problem was one of noise. The physical monitoring found a much higher incidence of this than the overall survey. In the test house a number of different ventilation scenarios were tested over a week. The bedroom with an ensuite performed reasonably well, but the bedroom which relied on a remote dMEV only achieved good ventilation when the windows were open at night. The next best scenario was when the occupants left the bedroom door open overnight. Subsequent modelling suggests that air flows from door undercuts are less effective. The findings would suggest that whilst there are some situations where a dMEV system can assist with the ventilation provision of modern airtight homes, the ability to act as a whole house system is limited, particularly in larger more complex layouts, and where ventilation loads are high. Although trickle ventilation provision in habitable rooms did not appear to be a major determinant of carbon dioxide concentrations in the monitored dwellings, these results should be interpreted with caution, given the small sample size and large number of confounding variables identified. It is likely therefore, that the impact of reduced area of trickle ventilation was overshadowed by other key components such as air flow pathways, pressure differentials, dMEV extract rates etc. As such, the system as a whole requires careful design, taking into account the house layout, paths for air movement (including undercuts and pass vents), the nature of the mechanical system, and consideration of remote rooms. The system will only be effective when these are optimised. Issues to consider therefore include: o Better design of ventilation strategies using dMEV as a component of the system and accounting for other key components o A need for pass vents between rooms, fire protected where required, ideally at higher levels o Better standards for commissioning and testing in use o Improved standards for noise for as-installed systems o Better design of occupant interfaces of mechanical systems, in particular boost modes and occupant control elements o Better advice and information for occupants about the ventilation system, its optimal use, and requirements for maintenance o Fall-back strategies for ventilation, where mechanical systems may fail or become sub-optimal over time o Direct extract ventilation for non-flued gas appliances o The development of performance standards for ventilation rates that can be compared with in-use data, and that provide an alternative means of complianc

Black race as a predictor of poor health outcomes among a national cohort of HIV/AIDS patients admitted to US hospitals: a cohort study

BACKGROUND: In general, the Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) population has begun to experience the benefits of highly active antiretroviral therapy (HAART); unfortunately, these benefits have not extended equally to Blacks in the United States, possibly due to differences in patient comorbidities and demographics. These differences include rates of hepatitis B and C infection, substance use, and socioeconomic status. To investigate the impact of these factors, we compared hospital mortality and length of stay (LOS) between Blacks and Whites with HIV/AIDS while adjusting for differences in these key characteristics. METHODS: The 1996-2006 National Hospital Discharge Surveys were used to identify HIV/AIDS patients admitted to US hospitals. Survey weights were incorporated to provide national estimates. Patients < 18 years of age, those who left against medical advice, those with an unknown discharge disposition and those with a LOS < 1 day were excluded. Patients were stratified into subgroups by race (Black or White). Two multivariable logistic regression models were constructed with race as the independent variable and outcomes (mortality and LOS > 10 days) as the dependent variables. Factors that were significantly different between Blacks and Whites at baseline via bivariable statistical tests were included as covariates. RESULTS: In the general US population, there are approximately 5 times fewer Blacks than Whites. In the present study, 1.5 million HIV/AIDS hospital discharges were identified and Blacks were 6 times more likely to be hospitalized than Whites. Notably, Blacks had higher rates of substance use (30% vs. 24%; P < 0.001), opportunistic infections (27% vs. 26%; P < 0.001) and cocaine use (13% vs. 5%; P < 0.001). Conversely, fewer Blacks were co-infected with hepatitis C virus (8% vs. 12%; P < 0.001). Hepatitis B virus was relatively infrequent (3% for both groups). Crude mortality rates were similar for both cohorts (5%); however, a greater proportion of Blacks had a LOS > 10 days (21% vs. 19%; P < 0.001). Black race, in the presence of comorbidities, was correlated with a higher odds of LOS > 10 days (OR, 95% CI = 1.20 [1.10-1.30]), but was not significantly correlated with a higher odds of mortality (OR, 95% CI = 1.07 [0.93-1.25]). CONCLUSION: Black race is a predictor of LOS > 10 days, but not mortality, among HIV/AIDS patients admitted to US hospitals. It is possible that racial disparities in hospital outcomes may be closing with time